We are interested in understanding the relative contribution, and the spatial/cellular specificity of the different neurosignaling pathways to the HDR elicited by functional brain stimulation. The working hypothesis is that increases in CBF are mediated by substances released by the parenchyma that act on the cerebral vessels and regulate their tone. The outstanding questions are: (i) which substances are released by what cells and under which circumstances? (ii) What is the spatial specificity of with respect to the cortical architecture? (a) The Role of COX-2: COX-2 is an enzyme involved in the conversion of arachidonic acid into substances generally known as prostanoids. In the brain, COX-2 is constitutively expressed and represents the primary isoform under physiological conditions. Involvement of COX-2 products in the neurovascular coupling has long been investigated and recent reports have shown that selective inhibition of COX-2 produces a 50% drop in the CBF response to somatosensory stimulation without affecting baseline CBF levels. However, these studies did not directly measure the neuronal activity following COX-2 inhibition, nor did they administer COX-2 pathway products, leaving open the questions (i) whether the observed attenuation in the CBF response resulted from a decrease in electrical activity or from a disruption of the cerebrovascular coupling;and (ii) whether this effect was reversible. We then hypothesized that: (i) COX-2 inhibition would produce a decrease in the hemodynamic response to functional brain stimulation without concomitant decreases in the neuronal response, thereby directly demonstrating the involvement of COX-2 in cerebrovascular coupling;and (ii) that this effect could be reversed by a systemic administration of a COX-2 derived vasodilatory product of arachidonic acid metabolism, prostaglandin E2 (PGE2). We tested these hypotheses in the alpha-chloralose anesthetized rat model of somatosensory stimulation. CBF and BOLD fMRI responses were measured before and during COX-2 inhibition with Meloxicam (MEL), a preferential COX-2 inhibitor and following a bolus of the major vasodilatory product of the COX-2 pathway, PGE2. The MEL treatment attenuated COX enzymatic activity by 57%, and imposed a progressive attenuation of the CBF response to somatosensory stimulation to 32% of the original pre-treatment response. We did not observe any effects of COX-2 inhibition on somatosensory evoked potentials (SEP), confirming the specific involvement of COX-2 in cerebrovascular coupling. When PGE2 was administered following COX-2 inhibition, we obtained a partial recovery of the CBF response to 52% of the original response, indicating a permissive role of PGE2 in cerebrovascular coupling. Similar results were obtained for the BOLD contrast. We observed a cortical laminar dependence to the CBF and BOLD responses in all pharmacological conditions. In agreement with earlier work, the CBF response prior to pharmacological perturbations was highest in layer IV. Continued administration of MEL caused a gradual attenuation of the CBF response across all layers. The maximum attenuation occurred in layer IV (P <0.001), essentially flattening the laminar profile of the CBF response. Administration of PGE2 restored the original laminar profile, indicating a preferential sensitivity of the HDR to COX-2 activity in layer IV. On the other hand, the BOLD laminar profile exhibited the maximum change in layer 1 most likely due to the effect of draining veins. While the BOLD profile was maintained throughout the pharmacological manipulation, there was also a trend of layer IV to be more affected than the other layers. The alteration of the cortical CBF profile by COX-2 manipulation has important implications to understanding cerebrovascular coupling and, in particular, to our research aims and goals. Recent immunohistochemistry studies of COX-2 distribution in rat neocortex have found that COX-2 was predominantly expressed in apical dendritic processes of pyramidal neurons in layers II-III and V, as well as in spiny dendrites and axonal terminals of excitatory neurons in layer IV, the latter receiving direct input from the thalamus via the lemniscal pathway. In further support of recent postulates of afferent driven blood flow control, the COX-2-mediated release of PGE2 may thus underlie an important thalamus-driven mechanism of local vasodilatation, providing a way for the microvasculature to follow the layer-specific flow of electrical activity in the cortex. It will be interesting to compare in further detail the cortical expression of COX-2 with the laminar profiles of the CBF response to increased neural activity. (b) The Role of NO: Another major neurosignalling pathway implicated in cerebrovascular coupling is the one that leads to production of nitric oxide (NO). In fact, due to its major function as a potent vasodilator, NO has been frequently interrogated as the most prominent signaling molecule in cerebrovascular coupling. Nitric oxide is synthesized endogenously by nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of L-arginine to L-citrulline and NO. Among the different NOS isoforms, the neuronal NOS (nNOS) has been found in a range of different brain regions, with nNOS rich axonal terminals frequently impinging on cerebral microvessels. Furthermore, nNOS cleaves NO from L-arginine in a Ca2+-dependent reaction and is physically anchored to the Ca2+-permeable NMDA-receptor (NMDA-R) by two postsynaptic density proteins, PSD-93 and PSD-95, and increased NO production after synaptic activity has been reported. Together, these characteristics make NO a prime candidate for a molecular messenger in the direct or neurogenic regulation of focal cerebral blood flow (CBF) following increases in the local neuronal activity. Previous studies in anesthetized rodent models reported significant, yet varying degrees of attenuation (by 30% to 90%) of the CBF response to somatosensory stimulation after NO inhibition. The wide range of attenuation reported likely resulted from differences in the pharmacological paradigms employed and the relative degrees of neuronal versus endothelial NOS inhibition achieved in addition to varying sensitivities of the different techniques for CBF estimation. At the same time, the effect of NOS inhibition on neuronal activity and thus neurovascular coupling remains unclear: some studies reported no concomitant changes in cerebral glucose metabolism or SEP, while others observed a 40% to 60% decrease in SEP amplitudes after NOS inhibition. To investigate the effect of the inhibition of neuronally derived NO on the spatial and temporal profiles of hemodynamic and neuronal responses to functional brain stimulation, using fMRI and SEP measurements in alpha-chloralose anesthetized rats before and after administration of a bolus of 7-nitroindazole (7-NI), a potent in vivo inhibitor of neuronal nitric oxide synthase (nNOS). We reported that administration of 7-NI caused a significant attenuation of the CBF, BOLD, and CBV responses to stimulation in the absence of any statistically significant effect on the resting perfusion and that at all times, the variation of the baseline perfusion was within the standard error of the CBF estimate, below 10%. Treatment of the rats with 7-NI caused complete suppression of the CBF response to somatosensory stimulation, the average across subject CBF response was 53% before treatment and it was nulled by 7-NI to -0.63%. Meanwhile, the average across subject BOLD response was reduced from 4.90% before 7-NI to 1.30% after 7-NI and the average across subject CBV response decreased from 41% before treatment to 4.60% after nNOS inhibition. SEP amplitudes were also affected, albeit to a lesser extent, by 7-NI administration.